DEVELOPMENT OF A SIMPLE TRANSIENT ASSAY FOR AC DS ACTIVITY IN CELLS OF INTACT BARLEY TISSUE/

The development of a barley (Hordeum vulgare L.) transformation system made it possible to consider the use of maize Activator/Dissociation (Ac/Ds) transposable elements for gene tagging in transgenic barley pl ants. However, barley transformation is time-consuming, and therefore a simple transient assay for Ac/Ds activity in intact barley tissues w as developed to test the components of a proposed gene tagging system, prior to their stable introduction into plants. in this assay, barley scutellar tissue is co-transformed with constructs containing the mai ze Ac transposase gene and an Escherichia coli uidA reporter gene (Gus ), the expression of which is interrupted by a maize Ds element. In tr ansformed barley scutellar cells, Ac transposase-mediated excision of the Ds element generates a functional Gus gene, leading to histochemic ally detectable GUS activity. Characterization of the excision product s showed that they had a pattern of nucleotide deletions and/or transv ersions similar to that found in maize and other heterologous plant sy stems. In addition, although contrary to the situation observed in het erologous dicot systems, efficient Ds excision in barley a heterologou s monocot system, appears to be inversely associated with Ac copy numb er, a finding similar to the Ac dosage effects observed in maize. The transient assay was used to demonstrate functional transposase activit y in barley callus lines stably transformed with an Ac transposase gen e.